Prevalence and Factors Associated with Opisthorchis viverrini Infection in Khammouane Province , Lao PDR

In many countries of South-East Asia infection with the liver fluke Opisthorchis viverrini (OV) is of considerable importance as a risk factor for cholangiocellular carcinoma (CC) development (Haswell-Elkins et al., 1994; Sriamporn et al., 2004; Sripa et al., 2011). In Viet Nam, Clonorchis sinensis may also be found (Doanh and Nawa, 2016). Infection with liver flukes (Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis) has been determined to be a class I carcinogen by the International Agency for Cancer Research evaluation committee (IARC, 1994). It has proved very difficult to change the dietary lifestyle habit of eating raw fish containing liver fluke eggs (Jongsuksuntigul and Imsomboon, 2003; Sripa et al., 2011) and a complicating factor is the presence of reservoir canine and feline hosts (Enes et al., 2010; Aunpromma et al., 2012). Furthermore, while the antihelminthic drug praziquantel may be used to control OV infection it maygive rise to an unwise dependency (Saengsawang et al., 2013) and repeated cycles of re-infection and treatment with praziquantel may increase the risk of CC (Kamsa-ard et al., 2015). As in neighbouring Thailand and Cambodia (Sithithaworn et al., 2012), OV has long been known in Laos (Bedier and Chesnau, 1929) and more recent reports have confirmed its presence as a common parasite in central and southern parts of the country ( Kobayashi et


Introduction
In many countries of South-East Asia infection with the liver fluke Opisthorchis viverrini (OV) is of considerable importance as a risk factor for cholangiocellular carcinoma (CC) development (Haswell-Elkins et al., 1994;Sriamporn et al., 2004;Sripa et al., 2011).In Viet Nam, Clonorchis sinensis may also be found (Doanh and Nawa, 2016).Infection with liver flukes (Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis) has been determined to be a class I carcinogen by the International Agency for Cancer Research evaluation committee (IARC, 1994).It has proved very difficult to change the dietary lifestyle habit of eating raw fish containing liver fluke eggs (Jongsuksuntigul and Imsomboon, 2003;Sripa et al., 2011) and a complicating factor is the presence of reservoir canine and feline hosts (Enes et al., 2010;Aunpromma et al., 2012).Furthermore, while the antihelminthic drug praziquantel may be used to control OV infection it maygive rise to an unwise dependency (Saengsawang et al., 2013) and repeated cycles of re-infection and treatment with praziquantel may increase the risk of CC (Kamsa-ard et al., 2015).
As in neighbouring Thailand and Cambodia (Sithithaworn et al., 2012), OV has long been known in Laos (Bedier and Chesnau, 1929) and more recent reports have confirmed its presence as a common parasite in central and southern parts of the country ( Kobayashi et

Design
The study design was a cross-sectional analytic study conducted in the Thakek district, Khammouane Province, Laos PDR, during the period June -July, 2015.

Subjects
There were 150 villages (88,229 population) in the Thakek district .10 villages were randomized to be study samples.There were 3,494 population aged 15 year-old and above in these 10 villages.A required sample size of 237 was estimated using formulae for proportion estimation (Lemeshow et al., 1991) and multiple logistic regression (Hsieh et al., 1998).Systematic random sampling was applied to select the study subjects.

Data collection
All stool samples were analysed for the presence of OV eggs using the Kato's thick smear technique, and participants were interviewed by a research assistant using a structured questionnaire to obtain demographic information, habit of defecation, smoking, alcohol consumption and record the reported consumption (yes or no) of unsafely prepared fish, defined as one of five types of dishes containing raw freshwater fish and one containing freshwater fish which was likely to have been inadequately fermented.All participants found to be infected by OV were provided with a treatment dose of praziquantel.

Statistical analysis and ethical approval
Descriptive statistics were used to summarise the data.Associations between OV infection status and potential risk factors were analysed using unconditional logistic regression.Variables significant at the p<0.25 level on univariate analysis were included in multivariate analysis with backward elimination.Additional candidate variables were factors which were non-significant in the univariate analysis, but which have been reported as strong risk factors for OV infection in previous studies.Statistical analyses were performed using Stata version 10.0 (Stata Corp, 2007).Statistical significance in the final model was set at p≤0.05.
The research was approved by the Khon Kaen University Ethics Committee for Human Research (reference no.HE582126).

Results
Overall, the proportion of individuals positive for OV infection was 54.8%.Table 1 shows the results of univariate and multivariate analyses of factors contributing to risk of infection with Opisthorchis viverrini.Age, education level, past use of praziquantel and defecation practice were not found to be risk factors.While alcohol

Discussion
The present study indicated that the infection rate of OV in our rural Laos community remains high and is directly related to gender and consumption of raw fish.The geographic pattern of OV infection is very uneven, but high rates are more likely in rural than urban environments, especially in wetlands and agricultural areas (Wattanayingcharoenchai et al., 2011).A common pattern that is emerging in each country of Mekong basin is the difference in transmission of OV between lowlands which have high prevalence versus highlands which have low prevalence (Sithithaworn et al., 2012).
In an earlier study in Laos, Champasack province, the rate was similar to that in the present study at 61.1% (Forrer et al., 2012).In Cambodia, while a low level of 4.6% was reported for Kratie Province (Sohn et al., 2012), in Takeo Province the percentage positive for OV was 46.4-50.6%(Yong et al., 2012).Four Cambodian provinces were identified as endemic areas of OV infection (Miyamoto et al., 2014).Reported rates in Thailand vary between 4.6% to 60.8% (Sithithaworn et al., 2012).High variability has also been reported between the different districts within provinces; for example, the age and gender adjusted proportion of the population infected in 20 districts of Khon Kaen Province varied between 10.0% and 70.9% (Sriamporn et al., 2004).Over the period 1981-2001 a variety of different surveys indicated that the Thai national prevalence of OV infection had fallen from 63.6% to 9.6%, but the most recent a data indicate the prevalence rate in the Northeast region remains high at 16.6% and had not decreased over the previous decade (Sithithaworn et al., 2012).In a review Kaewpitoon et al. (2015) reported 7 of 17 community-based surveys to have prevalences higher than 20%.and the highest was 45.7%.In upper Northeast Thailand, Nakhorn Phanom demonstrated a rate of 40.9% and four of seven provinces had a prevalence >20% (Thaewnongiew et al.,. 2014) .Rangsin et al. (2009) completed a two-year prospective survey of villagers of Chachoengsao Province in the Central region east of Bangkok and reported that the prevalence rate of OV infection increased from 21.3% to 26.2%.
However, it should be stressed that caution must be used in interpretation of data obtained by faecal egg examination.This method alone can lead to misidentification at the species level because of morphological similarity between the eggs of liver flukes and minute intestinal trematodes.In fact, recent surveys in Vietnam revealed that infection with several minute intestinal flukes, such as Haplorchis pumilio and H. taichui, are much more common than infection with C. sinensis or OV (Doanh and Nawa, 2016).Detection of OV-infection by urine OV-ES assay showed much greater diagnostic sensitivity and diagnostic specificity than the current "gold standard" FECT method for the detection and quantification of OV infection (Worasith et al., 2015) Immunodiagnosis of opisthorchiasis using parasite cathepsin F has been proposed (Teimoori et al., 2015) along with mitochondrial loop-mediated isothermal amplification (Le et al., 2012).PCR methods (tested in an experimental model (Wongratanacheewin et al., 2001), have been based on cytochrome c oxidase subunit one (cox1) and NADH dehydrogenase subunit one gene (nad1) (Buathong et al., 2015).Specific diagnosis of OV is also possible using loop-mediated isothermal amplification (LAMP) targeting copro-DNA (Arimatsu et al., 2012;2015).One other approach is to apply a faecal parasite concentrator (Kaewpitoon et al., 2016).Another is identification of plasma protein tyrosine phosphatase alpha and fibronectin associated with liver fluke (Khoontawa et al., 2012).Earlier, repeated stool sampling and use of multiple techniques was found to enhance the sensitivity of helminth diagnosis in Laos (Savasone et al., 2015).Also in Laos, use of a handheld micoscope has been advocated (Bogoch et al., 2016).
Regarding risk factors for infection, in Champasack province of Laos age, Lao Loum ethnicity, educational attainment, occupation (i.e., rice farmer, fisherman, and animal breeder), and unsafe drinking water source have been identified as risk factors for infection (Forrer et al., 2012).A history of praziquantel treatment, access to sanitation, and distance to freshwater bodies were found to be protective factors (Forrer et al., 2012).Infection was significantly associated with the children's experiences of eating raw fish, frequency of eating raw fish by their guardians and maternal educational and career in southern Laos (Tomokawa et al., 2013).In Laos there is a low degree of knowledge among local people on the health risks related to frequent consumption of raw or insufficiently cooked fish (Xayaseng et al., 2013).In Thailand a history of OV infection, a history of taking praziquantel, and unsafe disposal of waste food were predictive of infection in one study (Chudthaisong et al., 2015).In another in North-East Thailand, male gender was found to be significantly and positively associated with OV infection while education to secondary school level or above was a significant protective factor, whereas eating of unsafely prepared fish and knowledge about OV were not significantly related to infection status (Chaiputcha et al., 2015).Age and gender were also singled out as importnat (Saengsawang et al., 2012) while smoking and alcohol consumption were associated with an increased chance of acquiring OV infection (Yeoh et al., 2015).Thaewnongiew et al. (2014) reported factors related to opisthorchiasis to be (a) sex, (b) age (especially > 50), (c) proximity and duration living near a water body, and (d) eating raw and/or fermented fish.A significant positive association with OV prevalence was found for the combined land use classes of water and of farmland with high water content, sugesting the need to incorporate land use planning into control strategies (Wang et al., 2013).Clearly avoidance of the metacercariae in cyrpinoid fish in Khammouane province (Khemphavanh et al., 2009) is a high priority.How this is to be achieved will undoubtedly be a focus of continuing research.Whether the local population can be persuaded to adopt pla-som fermentation for more than four days and refrigerating fish for three days before pla-som processing which can prevent O. viverrini infection (Onsurathum et al., 2016) is one alternative which requires exploration, along with other community based-interventions.

Table 1 . Factors Associated with Opisthorchis Viverrini (OV) Infection on Univariate and Multivariate Analysis
:http://dx.doi.org/10.7314/APJCP.2016doi.org/10.7314/APJCP..17.3.1589Prevalence and Factors Associated with Opisthorchis Viverrini Infection in Lao PDR consumption and smoking were positive on univariate analysis this did not persist on multivariate analysis.Only gender and consumption of raw fish of type goi bplaa dip remained significant.